Heterologous Expression and Kinetic Characterization of Human Cytochromes P-450: Validation of a Pharmaceutical Tool for Drug Metabolism Research

• Published in: Drug metabolism and disposition Vol. 27; no. 10; pp. 1117 - 1122
• Main Authors: MASIMIREMBWA, C. M, OTTER, C, BERG, M, JÖNSSON, M, LEIDVIK, B, JONSSON, E, JOHANSSON, T, BACKMAN, A, EDLUND, A, ANDERSSON, T. B
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Pharmacology and Experimental Therapeutics 01.10.1999
• Subjects: Applied microbiology; Biological and medical sciences; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System - metabolism; Fundamental and applied biological sciences. Psychology; General pharmacology; Humans; Kinetics; Medical sciences; Medicin och hälsovetenskap; Microbiology; Miscellaneous; Pharmaceutical Preparations - metabolism; Pharmacokinetics. Pharmacogenetics. Drug-receptor interactions; Pharmacology. Drug treatments; Recombinant Proteins - metabolism; Saccharomyces cerevisiae; Substrate Specificity; Human; Drug; Validation; Unspecific monooxygenase; Enzyme; Isozyme; Cytochrome P450; Metabolism; In vitro; Research and development; Fungi; Drug-metabolizing enzyme; Ascomycetes; Models; Oxidoreductases; Recombinant protein; Saccharomyces cerevisiae; Thallophyta
• ISSN: 0090-9556; 1521-009X

Drug metabolism studies in the early phases of drug discovery and development will improve the selection of new chemical entities that will be successful in clinical trials. To meet the expanding demands for these studies on the numerous chemicals generated through combinatorial chemistry, we have heterologously expressed nine human drug-metabolizing cytochromes P-450 (CYPs) in Saccharomyces cerevisiae . The enzymes were characterized using known marker substrates CYP1A1/1A2 (ethoxyresorufin), 2C8 (paclitaxel), 2C9 (diclofenac), 2C19 ( S -mephenytoin), 2D6 (bufuralol), 2E1 (chlorzoxazone), and 3A4/3A5 (testosterone). All of the CYPs showed the expected substrate specificity except for chlorzoxazone hydroxylation, which, in addition to CYP2E1 and 1A2, was also catalyzed by CYP1A1 with a high turnover. The apparent Michaelis-Menten parameters obtained for each CYP were within the ranges of those reported in the literature using human liver microsomes and/or recombinant CYPs. The K m for CYP2E1-catalyzed chlorzoxazone hydroxylation was, however, much higher (177 μM) than that obtained using liver microsomes (40 μM). CYP-selective inhibitors, α-naphthoflavone (CYP1A1/1A2), quercetin (2C8), sulfaphenazole (2C9), quinidine (2D6), and ketoconazole (3A4/3A5) showed significant isoform-selective inhibitory effects. We have shown that ticlopidine is a potent inhibitor of CYP2C19 (IC 50 = 4.5 μM) and CYP2D6 (IC 50 = 3.5 μM) activities. We have therefore successfully set-up and validated an “in-house” heterologous system for the production of human recombinant CYPs for use in metabolism research.